The present invention relates to methods and compositions for the production of recombinant Adeno-Associated Viruses (rAAV). In particular, the invention discloses nucleic acid constructs and packaging cells having improved properties for rAAV production, as well as novel methods of titration and characterization of rAAV preparations. The invention also describes novel sequences which promote or increase the packaging of nucleic acids in rAAV, and their use for producing rAAV with high efficiency. The invention can be used for producing or testing high quality rAAV preparations, for biological, preclinical, clinical or pharmaceutical uses.
Wild type Adeno-Associated Virus (wtAAV) is a naturally defective parvovirus which requires co-infection with a helper virus, such as adenovirus or herpes virus, in order to establish a productive infection. The virus is not associated with any human disease and has been shown to have a broad host range of infection in vitro. AAV has a relatively simple genome organization composed of two major genes coding for the regulatory (rep) and structural (cap) proteins. Three viral promoters located at map unit 5 (p5), 19 (p19) and 40 (p40) control the synthesis of mRNA coding for the four Rep and the three Cap proteins. The viral genome is flanked by 145 bases inverted terminal repeats (ITRs) which contain palindromic sequences necessary in cis for replication of the viral genome (Leonard and Berns, 1994).
Recombinant AAV viruses (rAAV) are derived by deleting the rep and cap genes which are replaced by the transgene and the transcriptional control elements needed for its expression. The only viral sequences retained in cis are the viral ITRs (Muzyczka, 1992). The ability of rAAV to efficiently transduce tissues in mice such as the muscle, the retina or the liver (Fisher et al., 1997; Flannery et al, 1997; Kessler et al., 1996; Koeberl et al., 1997; Snyder et al., Herzog et al., 1997; 1997; Xiao et al., 1996; Zolotukhin et al, 1996) and to lead to a prolonged gene expression with little to no pathology makes this virus unique among the family of viral vectors. In this respect, rAAV can be used in vitro for recombinant protein production, gene regulation studies, AAV protein production (to be used in non-viral gene delivery systems), etc. rAAV can also be used ex vivo or in vivo, to deliver genes of interest for biological, toxicological, prophylactic or therapeutic indications for instance. In this regard, it should be noted that several clinical trials are currently ongoing using a rAAV gene delivery vector.
However, widespread use of rAAV is hampered by the relatively cumbersome and inefficient procedure needed to produce it at high titers and in sufficient amount and quality. The standard procedure relies upon the transfection of 293 cells with two plasmids: a plasmid providing in trans the rep and cap functions, and the rAAV vector plasmid itself. After subsequent infection with an adenovirus, rAAV particles are assembled in the nuclei of the cells concomitantly with adenoviral particles. rAAV stocks are obtained after purification from total cell lysates through CsCI gradients (Snyder et al., 1996).
However, these methods represent relatively long and complex procedures, which cannot be easily scaled-up. Furthermore, because of the number of constructs required, recombination events have been observed leading to rAAV preparations which are contaminated with replicating AAV particles and with adenoviruses. There is therefore a need for improved methods of producing rAAV, for biological, preclinical, clinical or pharmaceutical uses. In particular, there is a need for methods of producing rAAV preparations with high titers of infectious particles and which are essentially free of adenoviruses. There is also a need for methods to produce rAAV preparations with significantly reduced contamination by replication competent or recombined AAV. There is also a need for improved methods of titration of rAAV preparations, and for methods of characterization of such preparations, i.e., for use in Quality Control steps, as well as for detecting rAAV or contaminants in biological fluids, for instance.
The present invention now provides novel methods and compositions for producing and characterizing rAAV preparations. In particular, the invention provides methods of producing rAAV with very high yields of infectious particles and essentially free of detectable adenovirus contamination.
The present invention relates to compositions and methods for producing and/or characterizing rAAV preparations of improved quality. The invention relates more particularly to improved nucleic acid constructs that provide for an efficient production of rAAVs, as well as to compositions, cell lines and methods for characterizing rAAV preparations. The invention can be used to produce rAAV for biological, preclinical or clinical uses, e.g., pharmaceutically acceptable rAAV preparations.
Within the context of the present invention, a recombinant Adeno-Associated Virus (rAAV) designates an AAV virus which comprises at least a recombinant nucleic acid genome. More specifically, rAAVs generally comprise a recombinant genome lacking a functional rep and/or cap region, and comprising a heterologous nucleic acid. Most conventional rAAVs comprise a recombinant genome lacking the entire Rep and Cap regions, which are replaced by the heterologous nucleic acid. Such recombinant genomes thus usually comprise the heterologous nucleic acid flanked by the left and right Inverted Terminal Repeats of AAV. rAAVs may comprise additional modifications, such as artificial or heterologous capsid proteins, for instance. Furthermore, the recombinant genome may comprise, in replacement or in addition to ITR sequence, RES elements as disclosed in the present invention. rAAVs may be derived from different serotypes of AAV, such as for instance AAV-2, AAV-3, AAV-4 or AAV-6.
In the present invention, except otherwise indicated, all references to nucleotide positions of the AAV genome are made with respect to the sequence of wild-type AAV-2 available at Genebank under number AF043303.
A recombinant Adeno-Associated Virus vector plasmid (rAAV vector plasmid) designates a nucleic acid construct comprising a copy of the genetic information to be packaged into AAV capsids, to form the rAAV. The rAAV vector plasmid is therefore any nucleic acid construct comprising the recombinant genome of the rAAV as defined above, preferably a heterologous nucleic acid flanked by one or two AAV Inverted Terminal Repeats (ITR) and/or, optionally, one or several RES elements. The rAAV vector plasmid can be autonomously replicating, conditionally replicating, or stably integrated into the genome of the packaging cell.
A rep-cap plasmid designates any nucleic acid construct encoding the rep and/or cap proteins, which provide in trans the AAV complementing functions lacking in the rAAV vector plasmid. Generally, the rep-cap plasmid encodes Rep78, Rep68, Rep52, Rep40, VP1, VP2 and VP3. The rep-cap plasmid can be a single construct encoding all the required REP and CAP proteins, under the control of the same or separate promoters. The rep-cap plasmid can also be a mixture of distinct nucleic acid constructs encoding one or several REP and CAP proteins. The rep-cap plasmid can be autonomously replicating, conditionally replicating, or stably integrated into the genome of the packaging cell.
As indicated above, a first aspect of the invention resides in a method of characterizing rAAV preparations or stocks. Indeed, because of the complex methods and constructs needed to produce rAAV, it is important to have efficient and accurate methods of characterizing the rAAV preparations obtained, especially for biological uses. Previous methods known in the art essentially focus on the determination of the contamination by adenoviruses, and/or the number of infectious AAV particles. Also, in determining the number of infectious rAAV particles, most prior art methods rely on the detection of the expression product of the nucleic acid inserted in the rAAV genome and are therefore transgene-dependent. The invention now provides a new method of characterizing rAAV preparations, which is transgene-independent, sensitive, accurate, and allows the measure of adenovirus and recombined AAV contaminants. This method can be used in any rAAV production method, or as a Quality Control in biological processes, to check the quality of a preparation and, optionally, allow the improvement of the production parameters.
More specifically, an object of the present invention is a method of characterizing a rAAV preparation, said method comprising:
a) contacting a sample of said preparation with a culture of cells expressing the rep proteins,
b) contacting a sample of said preparation with a culture of cells expressing the rep proteins, co-infected with an adenovirus, and
c) contacting a sample of said preparation with a culture of cells which do not express Rep proteins, co-infected with an adenovirus,
and measuring the presence of viruses in cultures a), b) and c).
As will be discussed in more details below, the rAAV preparation can be any preparation of rAAVs produced by any method. It is, preferably, a purified rAAV stock obtained from a rAAV producing cell culture extract. The rAAV preparation can be for instance a stock of rAAVs, to be assayed before administration to a mammalian, including a human being, for clinical or pharmaceutical purposes. In this regard, xe2x80x9ccharacterizingxe2x80x9d means within the context of the above method, determining both (i) the number of infectious rAAV particles and (ii) the presence of contaminating viruses, in particular contaminating adenoviruses and rep-positive rAAVs in the preparation.
In the characterization method of this invention, the sample being contacted with the above indicated cell cultures can be a pure or a diluted sample of the rAAV preparation. In a preferred embodiment, serial dilutions of the rAAV preparation are being used, comprising for instance from about 5.104 to 50 infectious particles/ml.
For carrying out the claimed method, different cell cultures can be used. Preferably, of course, the cells are permissive to AAV, i.e., can replicate the AAV genome in appropriate conditions (i.e., in the presence of adenoviral helper functions). Suitable cell cultures include culture of human primary or established cell lines, preferably established cell lines; other mammalian cell lines or cultures, including canine or murine cells. Example of cells which can be used include for instance human cells such as nervous cells, fibroblasts, hepatocytes, myoblasts or the like preferably established as cell lines. More preferred cell lines include the HEK cells, Hela cells, Huh7, HT1080, J82 or T98G, for instance.
In conducting the method of this invention, it is preferred to use in all three contacting tests the same cell type (i.e., HT1080, Huh7, HeLa cells, etc.). In a preferred embodiment, the cell culture used in a), b) and c) is a culture of HeLa cells.
In an even more preferred embodiment, the cell cultures used in a) and b) are cultures of the same cell populations. More preferably, the cell culture used in c) is also essentially identical to those used in a) and b), except for the Rep status.
As explained above, the cells used in a) and b) express the Rep proteins, i.e. the proteins of AAV which are involved in the replication of the genome. The Rep region of AAVs produces essentially 4 major proteins, Rep78, Rep 68, Rep 52 and Rep 40. All these proteins are involved in the replication of AAVs and should be present in the cell culture, to ensure maximum efficacy. Accordingly, in a particular embodiment, the cells expressing AAV rep proteins used in the invention are cells which express Rep78, Rep 68, Rep 52 and Rep 40. Preferably, the Rep proteins are expressed under the control of the AAV p5 and p19 promoters. In a more particular embodiment, the cells express the AAV Rep proteins encoded by nucleotides 190-2278 of the AAV genome. Alternative embodiments use cells which express only some of the AAV rep proteins, such as Rep78/Rep68, for instance, which are known to be essential for replication, or any other combination thereof which allows replication of AAV genome.
In addition, in the method of this invention, it is preferred to use cells which also express AAV cap proteins, i.e., the proteins of AAV involved in the formation of the capsid. The Cap proteins (VP1, VP2 and VP3) are encoded by nucleotides 1850-4484 of AAV. These proteins can be expressed under the control of the natural AAV p40 promoter, or any heterologous promoter. Preferably, cells used in a) and b) also express the AAV Cap protein expressed by nucleotides 1700-4484 of the AAV genome.
In a variant, the invention relates to a method of characterizing a rAAV preparation, said method comprising:
a) contacting a first sample of said preparation with a culture of mammalian cells expressing the AAV rep proteins encoded by nucleotides 190-2278 of the AAV genome,
b) contacting a second sample of said preparation with another culture of the cells of a), co-infected with an adenovirus, and
c) contacting a third sample of said preparation with a culture of mammalian cells which do not express Rep proteins, co-infected with an adenovirus,
and measuring the presence of viruses in cultures a), b) and c).
More preferably, the mammalian cells in a) and b) above also express the AAV Cap proteins encoded by nucleotides 1850-4484 of the AAV genome. In a particularly preferred embodiment, the mammalian cells used in a) and b) comprise a nucleic acid sequence, integrated in their genome, which codes for the Rep and Cap proteins of AAV. The nucleic acid has for instance the sequence of nucleotides 190-4484 of the AAV genome. More preferably, the mammalian cells in a), b) and c) are HeLa cells, such as cells HeLaRC32, disclosed in the Examples.
In assays b) and c) of the instant method, the cell cultures are co-infected with an adenovirus. Usually, the adenovirus is of the group C, even more preferably of the serotype Ad2, Ad5, Ad7 or Ad12. Other types of adenoviruses can be used, such as for instance canine adenoviruses (CAV-2) which are known to complement AAV replication. Furthermore, the adenovirus can be wild-type or modified, in particular temperature sensitive. The doses of adenoviruses used can be adapted by the skilled artisan, depending on the cell types, the rAAV preparation, and the nature of the adenovirus. Generally, the adenovirus is used at an MOI of between 5 and 1000, preferably below 800, more preferably between 50 and 600. Finally, the adenovirus can be replaced with an adenoviral plasmid, i.e. a plasmid or combination of plasmids encoding the adenoviral functions necessary for AAV replication, although the use of a virus is preferred.
After the contacting of the rAAV preparation with the cell cultures, the presence of viruses is determined. In this regard, the measuring comprises measuring the presence of rAAV viruses within each test a), b) and c). More preferably, the measuring of the presence of viruses in cultures a), b) and c) comprises measuring (i.e., detecting) the presence of rAAV replicating DNA in the cells. This measure is usually accomplished by using rAAV-specific probes, optionally after amplification of the cellular nucleic acids with rAAV-specific primers. In a particular embodiment, a probe is used which is complementary to all or part of the rAAV genome, in particular to all or part of the heterologous nucleic acid present in the rAAV genome. The probe comprises preferably at least 100 bp, to ensure higher selectivity. The probe can be labeled with any conventional technique (enzymatic, fluorescent, radioactive, etc.). In a preferred embodiment, the detection of the presence of rAAV replicating DNA in the cells of a), b) and c) is performed after cell lysis without amplification of the cellular nucleic acids. Detection without amplification allows a better quantitative evaluation of the number of rAAV genomes present within the cells. Of course, amplification can be performed, prior to the detection, by using primers specific for regions of the heterologous nucleic acid present in the rAAV genome.
The detection of rAAV DNA within the cells is preferably accomplished by hybridization of the cellular nucleic acids with a probe as defined above. Preferably, the hybridization is performed on a solid support, such as a membrane, filter, etc., onto which the cellular nucleic acids have been transferred or onto which the cells have been directly lysed. The hybridization conditions can be either medium or, preferably, of high stringency. Typical hybridization conditions under high stringency are as follows: Dextran sulfate 5%, SSC 5%, SDS 0.1% liquid block 10%, (Amersham). The support is prehybridized 30xe2x80x2 at 65xc2x0 C., and the denatured probes are then hybridized overnight at 65xc2x0 C. It should be understood that the hybridization conditions can be adjusted, for instance by reducing the temperature or the washing conditions, by the skilled artisan.
Generally, the measure is performed 12-96 hours after the contacting, preferably less than 48 hours. Typically, when measuring comprises measuring the AAV replicating nucleic acids within the cell culture, the measuring is performed at about 24 hours post-contacting. It allows replication, but no release of AAV which could infect other cells.
The above method is particularly advantageous since it provides not only the titer of a preparation in infectious particles, regardless of the nature of the heterologous nucleic acid contained in the vector, but also the level of contamination by adenoviruses and rep-positive AAVs.
Indeed, the contacting with cell culture a), i.e., with a cell culture expressing rep proteins but in the absence of helper adenovirus functions, allows the identification of the presence of complementing adenoviruses within the rAAV preparation, i.e., the presence of contaminating adenoviruses. Indeed, it is known that AAV cannot replicate their genome in the absence of helper adenovirus functions. In this cell culture a), the replication of rAAV implies the presence of helper adenoviral function in the medium, i.e., of contaminating adenoviruses within the preparation. The number of cells in which rAAV is replicating can be directly correlated to the level of adenovirus contamination.
The contacting with cell culture b), i.e., with a cell culture expressing rep proteins, in the presence of helper adenovirus functions, allows the titration of infectious rAAV particles present in the preparation.
The contacting with cell culture c), i.e., with a cell culture which do not express rep proteins, in the presence of helper adenovirus functions, allows the identification of the presence of rep-positive AAV viruses within the rAAV preparation, i.e., the presence of contaminating rAAV which contain a rep-encoding nucleic acid or a rep protein.
The method is therefore very efficient and provides immediate information regarding the quality of a rAAV preparation, as illustrated in the Examples. Another object of the present invention therefore lies also in a method as described above, for detecting the presence of rAAV and/or rep-positive AAVs and/or adenoviral particles in biological fluids, in particular after in vivo administration of rAAV preparations in animals and/or human subjects. Such biological fluids are, more particularly, serum, urine, stool, saliva, broncho alveolar fluids, etc. The method is performed as described above, by contacting, in tests a), b) and c), samples of the biological fluids or dilutions or concentrates or derivatives thereof. It can thus be used to monitor safety issues during preclinical, clinical or pharmaceutical settings.
Furthermore, the above method allowed the inventors to establish improved conditions and improved nucleic acid constructs for producing rAAV stocks. Moreover, in performing the above characterization method, the inventors discovered the presence of rep-positive AAVs within the preparation, which led them to try to understand the molecular mechanisms responsible therefor. Indeed, because the nucleic acid constructs used in the production method are essentially free of overlapping regions between each other, homologous recombination events are essentially impossible. Non-homologous recombination events between the rep-cap plasmid and the rAAV vector plasmid might account for the presence of such rep-positive rAAVs in the final preparation. Surprisingly, the applicants found that the presence of rep-positive particles could be the result of an ITR-independent packaging of rep nucleic acid. Surprisingly, the inventors have now discovered the existence of non-ITR packaging regions within the AAV genome. Both the improved method and non-ITR packaging regions are also encompassed by the present application, as will be discussed below.
The invention therefore relates also to a method of producing rAAV preparations, comprising:
a) producing rAAVs in a cell culture expressing the Rep and Cap functions and the adenovirus helper functions, and
c) characterizing the rAAVs produced according to the method disclosed above.
More preferably, the method comprises:
a) producing rAAVs in a cell culture expressing the Rep and Cap functions and the adenovirus helper functions,
b) purifying the rAAVs produced, and
c) characterizing the rAAVs produced according to the method disclosed above.
The production of the rAAVs can be performed according to various methods, including conventional methods known in the art. For instance, step a) can be accomplished by co-transfection of a cell culture with a Rep-Cap plasmid, the rAAV vector plasmid and a helper adenovirus. Step a) can also be performed using a culture of cells which contain, stably integrated into their genome, nucleic acid construct(s) encoding the Rep and/or Cap proteins. Also, the helper adenovirus can be either wild-type adenovirus, or a replication deficient adenovirus, such as a E1-deficient adenovirus, for instance. In this embodiment, the cells used preferably produce the function(s) lacking in the adenovirus. In particular, the 293 cells are frequently used, which express the E1 functions of adenoviruses.
In a particular embodiment, in the producing step a), a rep-cap plasmid is used, which lack any functional ITR region, in particular any adenoviral ITR region. In this respect and, contrary to previous observations, the inventors have now shown that the use of a rep-cap plasmid containing the adenoviral ITR regions does not increase the yield of infectious particles. It is therefore a preferred embodiment of this invention to use a Rep-Cap plasmid lacking adenoviral ITR regions. Such plasmids are disclosed in the examples. In particular, a preferred Rep-Cap plasmid within the context of the instant invention is a plasmid containing a Rep-Cap unit consisting of residues 190-4484 of the AAV genome or fragments thereof encoding functional Rep and Cap proteins. Furthermore, the plasmids used may contain the homologous transcriptional promoter regions of the AAV Rep and Cap genes (i.e., promoters p5, p19 and p40), or any heterologous promoter region. Particular examples of such plasmids are, for instance:
plasmid pspRC, which contains the ITR-deleted AAV genome position 190-4484 (FIG. 1), and
plasmid pspRCC, which contains the rep gene (190-2278 bp of wtAAV) followed by the bGH polyadenylation signal and the CMV promoter leading the expression of the cap ORF (1882-4484 bp of wtAAV).
In this regard, a particular object of this invention also resides in plasmids pspRC and pspRCC.
In another particular embodiment of this invention, step a) comprises the co-transfection of a cell culture with a rep-cap plasmid, a rAAV vector plasmid and a helper adenovirus.
In a further particular embodiment of the present invention, step a) comprises the use of adenovirus helper plasmids, instead of a helper adenovirus. Indeed, as indicated before, while the production of rAAV requires the presence of adenovirus helper functions, the use of a helper adenovirus generally leads to a contamination of rAAV preparations by adenoviruses (defective, replicating and/or wild-type). In order to avoid such a contamination, the inventors have now found that adenovirus plasmids can be used, without significantly affecting the yields of rAAV produced. In this respect, particular plasmids can be used, such as plasmids carrying the entire adenoviral genome or portions thereof, which are sufficient to supply the functions required for rAAV production. In a particular embodiment, a plasmid is used carrying the entire adenovirus genome. Such a plasmid is for instance pAdc disclosed on FIG. 1. In another particular embodiment, a plasmid is used, which contains the adenoviral genome lacking the left and right ITRs and the packaging region. In a further preferred embodiment, a plasmid is used, which contains a defective adenoviral genome lacking the left and right ITRs, the packaging region and the E1 region. Such a plasmid is, for instance plasmid pAdxcex94 (FIG. 1).
Surprisingly, the inventors have now shown, for the first time, that the use of such plasmids in replacement of a helper adenovirus does not reduce the yields of infectious rAAV particles produced. Furthermore, the use of such plasmids avoids the production of contaminating adenoviruses in the preparations, as demonstrated in the examples. This embodiment thus represents a preferred way of carrying out the instant invention.
In this regard, a particular object of this invention also resides in plasmids pAdc and pAdxcex94.
Another object of this invention also resides in a method of producing rAAVs comprising:
(i) co-transfecting a cell culture with:
a rAAV vector plasmid,
a Rep-Cap plasmid devoid of ITR, containing preferably a Rep-Cap unit consisting of residues 190-4484 of the AAV genome or fragments thereof encoding functional Rep and Cap proteins, and
an adenovirus plasmid containing the entire adenoviral genome or a genome lacking the left and right ITRs, the packaging region and, optionally, the E1 region, and
(ii) recovering the rAAV produced.
In another particular embodiment of the present invention, step a) above comprises the use of a culture of cells which contain, in their genome, nucleic acid construct(s) encoding the rep and/or cap functions, preferably the rep and cap functions. In this embodiment, the production step a) comprises the co-transfection of this cell culture with the rAAV vector plasmid and with a helper adenovirus or an adenovirus plasmid as described above. This embodiment is advantageous in that it avoids the need for a Rep-Cap plasmid. Suitable cells for carrying this embodiment include any cells encoding the AAV REP and CAP proteins encoded by nucleotides 190-4484 of the AAV genome. These cells can be derived, as disclosed above, from human cells, such as embryonic cells, or even from other mammalian cells. Preferred cells are obtained from 293 cells, HeLa cells, A549 cell, Huh7 cells, HT1080, J82, T98G or HER cells. A specific example is the HeLaRC32 cells disclosed above, for instance.
Another object of this invention therefore resides also in a method of producing rAAVs comprising
(i) co-transfecting a culture of cells which contain, in their genome, nucleic acid construct(s) encoding the rep and/or cap functions, preferably the rep and cap functions, with:
a rAAV vector plasmid, and
a helper adenovirus or an adenovirus plasmid containing the entire adenoviral genome or a genome lacking the left and right ITRs, the packaging region and, optionally, the E1 region, and
(ii) recovering the rAAV produced.
Step b) of the above production method comprises the purification of the rAAV produced. Said purification can be performed according to various techniques, including methods known in the art such as centrifugation, clarification, and cesium chloride gradient purification. In this regard, the CsCl purification procedure disclosed in the art essentially comprises the centrifugation of the rAAV cell extract at 41 000 rpm for 48 hours (rotor sw41). The inventors have now shown that it is possible to significantly reduce the length of the centrifugation step, when the other parameters are adjusted. In particular, the inventors have now shown when the CsCl centrifugation is maintained for 6 hours at between 60 000 and 70 000 rpm, preferably between 65 000 and 70 000 rpm, the same level of purity is obtained than with previous methods, requiring 48 hours centrifugation.
A preferred embodiment of the invention therefore comprises centrifuging the rAAV preparation in a cesium chloride gradient for less than 12 hours, at between 60 000 and 70 000 rpm, preferably between 65 000 and 70 000 rpm.
Other purification methods can be used such as, in particular, chromatographic techniques. In this respect, a particular purification method uses anion exchange chromatography, optionally combined to exclusion chromatography. A specific purification protocol comprises for instance the loading of the rAAV preparation on an anionic exchange chromatography column, such as for instance Resource Q (Amersham, Pharmacia Biotech), followed by an exclusion chromatography, for instance onto a Sephacryl (S500) column, to exclude the viral particle and separate them out of residual protein and lipid contaminants.
A particular object of the present invention therefore also lies in a method of purifying rAAVs from a biological sample, comprising treating said sample in a cesium chloride gradient centrifugation at between 60 000 and 70 000 rpm, preferably for less than 12 hours, and recovering the fraction(s) containing the purified rAAVs.
Another particular object of this invention lies in a method of purifying rAAVs from a biological sample, comprising treating said sample at least by anion exchange chromatography and exclusion chromatography.
Furthermore, as indicated above, the invention also relates to novel nucleic acids with packaging activity. Indeed, the inventors have now discovered that nucleic acid regions, distinct from the ITRs, can mediate and/or increase the packaging of nucleic acids within AAV capsids. These regions, termed xe2x80x9cReplication Encapsidation Sequencexe2x80x9d or xe2x80x9cRESxe2x80x9d as well as their use for producing rAAVs, represent another particular object of the instant invention.
More particularly, within the context of the present invention, a xe2x80x9cReplication Encapsidation Sequencexe2x80x9d represents a sequence different from an AAV ITR sequence, which promotes and/or facilitates the packaging of a nucleic acid into an AAV capsid. Preferably, a RES element comprises a Rep Binding Element (RBE), where Rep78/Rep68 proteins can bind, and a terminal resolution site (trs), for the binding of endonucleases. Even more preferably a RES comprises a RBE site, a trs site and a palindromic sequence. Surprisingly, the Applicants have now found that regions favoring packaging of a nucleic acid into an AAV particle are present in the genome of viruses, such as AAV. These regions are distinct from the ITRs, and can promote the packaging of an ITR-free nucleic acid into an AAV particle. The invention therefore discloses new nucleic acids with packaging activity, that can be used to package nucleic acids into AAV capsids, or to improve the packaging efficiency of conventional rAAV vectors.
A particular object of this invention lies in an isolated Replication Encapsidation Sequence, wherein said RES is a nucleic acid sequence distinct from an AAV ITR sequence, which provides or promotes the packaging of a nucleic acid operably linked thereto into an AAV particle.
Preferably, a RES of this invention comprises a Rep Binding Element and a terminal resolution site, and, optionally, a palindromic sequence. A preferred RES according to this invention is an isolated nucleic acid, wherein, said nucleic acid provides or promotes the packaging of a polynucleotide operably linked thereto into an AAV particle.
A particular example is a RES comprising a region having the sequence of SEQ ID NO: 1 (GCC CGA GTG AGC ACG CAG) or a functional variant thereof.
More preferably, a RES of this invention comprises a region having the sequence of SEQ ID NO: 1 or a functional variant thereof and further comprises a region having the sequence of SEQ ID NO: 2 (GCG ACA CCA TGT GGT CAC GC) or a functional variant thereof.
A particular RES of this invention is a nucleic acid comprising SEQ ID NO: 3 (GCC CGA GTG AGC ACG CAG GGT CTC CAT TTT GAA) or a functional variant thereof, which nucleic acid having a packaging activity.
Even more preferably, a RES of this invention comprises SEQ ID NO: 3 or a functional variant thereof and SEQ ID NO: 2 or a functional variant thereof.
The term xe2x80x9cfunctional variantxe2x80x9d means any sequence comprising one or several structural modifications, that still retain the activity of the sequence, i.e., the binding to Rep78 or Rep68 for SEQ ID NOS: 1 and 3, and the ability to form a hairpin structure for SEQ ID NO: 2. More preferred functional variants retains at least 50% identity with the sequence disclosed in the examples. Particular examples of functional variants are sequences with one or several mutations, additions or deletions in the above sequences, in particular 1, 2 or 3 mutations.
Other typical variants are sequences which hybridize with the above sequences and retain the RES activity. To search for sequences in the genome of other viruses having some homologies with the RES fragment and be considered as variants, hybridization in low stringency conditions using the RES fragment as a probe are performed. As an example, hybridization is done overnight at 56xc2x0 C. instead of 65xc2x0 C. in the hybridization solution recommended by the manufacturer""s instructions (Amersham), which is 5xc3x97SSC, 0.1% (W/V) SDS, 5% (W/V) Dextran sulphate and 1/20 of liquid block. Washes are performed also in low stringency conditions, for instance: 1xc3x97SSC, 0.1% SDS at 56xc2x0 C. The RES can be artificial, semi-synthetic, viral or of any other origin.
Other functional variants are RES sequences derived from other parvoviruses which exhibit the functional properties of the above RES sequences.
Preferably, RES elements contain less than 400 bp. A specific example is provided on FIG. 8A, which consists of nucleotides 190-540 of AAV genome (i.e., 350 bp). Fragments of this RES are presented in FIG. 10, which represent particular embodiments of this invention, such as nucleotides 190-350, 230-350, 230-300, 250-300 or functional variants thereof, for instance.
As indicated above, the RES elements of this invention can be used either to promote the packaging of a nucleic acid into an AAV capsid, or to improve the packaging efficiency of rAAV vector plasmids. Accordingly, a particular object of this invention is a nucleic acid consisting of one or several RES elements fused to a heterologous polynucleotide lacking a functional ITR sequence.
The heterologous polynucleotide lacking a functional ITR sequence can be any nucleic acid sequence of interest, such as a nucleic acid coding for a protein or RNA of interest for instance. Fused means that the two elements are operably linked together, in the same or reverse orientation. The RES elements can be fused at the 5xe2x80x2 or 3xe2x80x2 ends of the heterologous nucleic acid, or inserted within said polynucleotide. Examples of such nucleic acids include for instance plasmid RES+CMVxe2x88x92LacZ or plasmid RESxe2x88x92CMV-LacZ as shown on FIG. 9. Any similar construct in which the CMV promoter is replaced with another promoter and/or the LacZ gene is replaced with another gene can be produced by the skilled artisan.
Another object of this invention is a rAAV vector plasmid, comprising a recombinant AAV genome and one or several RES element. A particular construct is, for instance, a plasmid comprising the following operably linked elements: 5xe2x80x2-ITR-RES-heterologous polynucleotide-ITR-3xe2x80x2.
The invention also relates to a rAAV particle comprising a recombinant nucleic acid genome, wherein said recombinant nucleic acid genome comprises one or several RES elements.
The invention can be used to produce rAAV and to characterize rAAV preparations, for use in various technical areas, such as experimental biology, preclinical studies, clinical studies or pharmaceutical indications.
Other advantages and uses of the present invention be disclosed in the following experimental section, which should be regarded as illustrative and not limitative.
Legend to the Tables
TABLE 1. Characterization of the 18 large scale rAAV stocks. Each vector was produced from 25 15-cm plates of 293 cells except for stocks marked with an asterisk which were produced from 50 15-cm plates of cells. The vector name indicates the promoter and the transgene inserted between AAV ITRs. The second column indicates the rAAV size (ITR to ITR); the third, the rep-cap construct used for the production and the fourth whether adenovirus (wtAd5 or Ad.dts) or an adenoviral plasmid (pAdc) was used. The rAAV titer was measured by: 1 dot blot, 2 RCA. Contaminations3 with adenovirus and rep-positive AAV particles were also measured by RCA. The last column indicates the final volume of virus after CsCl gradient purification and dialysis. rAAV stocks listed below the darker line in the middle of the table were purified following the centrifugation conditions described in Materials and Methods. Those listed above were purified following the protocol described by Snyder et al (1996). p./ml: particles/ml; i.p./ml: infectious particles/ml.
TABLE 2. Evaluation of different rep-cap expression plasmids for rAAV production. rAAV was produced from two 15-cm plates of 293 cells transfected with the AAVCMVnlsLacZ vector, the adenoviral plasmid pAdc and the indicated rep-cap construct. Cells were collected three days after transfection and cell extracts purified as described in Materials and Methods. Each stock was tittered by: 1 dot blot, 2 RCA and 3 by an LFU assay performed on HeLa cells. The final volume of virus was of 7 ml for each stock.
TABLE 3. Comparison of the two adenoviral plasmids pAdc and pAdxcex94 for rAAV production. rAAV was produced from two 15-cm plates of 293 cells transfected with the pspRC construct, the AAVCMVnlsLacZ vector and the indicated adenoviral plasmid. Cells were collected 72, 96 and 120 hours after transfection and cell extracts purified as described in Materials and Methods. Each stock was tittered by: 1 dot blot and 2 RCA.
TABLE 4. Measure of infectious and transducing rAAVCMVnlsLacZ particles produced using either Ad.dl324 (vAd) or an adenoviral plasmid (pAdc). Rep-cap functions were provided by the pspRC construct. In the case of the stock obtained with vAd, the virus was produced from 20 15-cm plates of 293 cells and the final volume was of 6.8 ml. In the case of the stock obtained with pAdc, the virus was produced from 50 15-cm plates of 293 cells and the final volume was of 13.4 ml. Recombinant AAV was tittered by: 1 dot blot, 2 RCA, 3 and 4 LFU assay on HeLa cells in the presence or in the absence of wtAd 5. ND, not done.